Landscape of potential germline pathogenic variants in select cancer susceptibility genes in patients with adult‐type ovarian granulosa cell tumors

Abstract Objective The objective of this study was to assess the frequency of potential germline pathogenic variants that may contribute to risk of development of adult granulosa cell tumors (AGCT) given the paucity of germline testing guidelines for these patients. Methods This was a retrospective cross‐sectional study analyzing comprehensive genomic profiling (CGP) results of AGCT with the FOXL2 p.C134W mutation submitted to Foundation Medicine between 2012 and 2022. Cases with a potential germline pathogenic variant were identified by filtering single nucleotide variants and short indels by variant allele frequency (VAF) and presence in ClinVar for select cancer susceptibility genes. Odds ratios for AGCT risk were calculated compared to a healthy population. Results Prior to analysis, 595 patients were screened and 516 with a somatic FOXL2 p.C134W mutation were included. Potential germline pathogenic variants in a DNA repair‐related gene (ATM, BRCA1, BRCA2, CHEK2, PALB2, PMS2, RAD51C, or RAD51D) were found in 6.6% of FOXL2‐mutated AGCT. Potential germline pathogenic CHEK2 variants were found in 3.5% (18/516) of AGCT patients, a rate that was 2.8‐fold higher than Genome Aggregation Database non‐cancer subjects (95% CI 1.8–4.6, p < 0.001). The founder variants p.I157T (38.9%, 7/18) and p.T367fs*15 (c.1100delC; 27.8%, 5/18) were most commonly observed. CHEK2 VAF indicated frequent loss of the wildtype copy of the gene. Conclusions These results support ongoing utilization of genomic tumor profiling and confirmatory germline testing for potential germline pathogenic variants. Further prospective investigation into the biology of germline variants in this population is warranted.


| INTRODUCTION
Adult granulosa cell tumors (AGCT) are ovarian sex cord-stromal tumors that occur at the site of female steroidogenesis.The need for improved biological understanding and treatment approaches is underscored by the fact that 50%-80% of patients die from recurrent disease.
2][3] A recent study by Hillman et al. reported that FOXL2 p.C134W was found in almost all of 423 patients' AGCTs sequenced by Foundation Medicine, but few currently actionable alterations were identified, emphasizing the critical need for precision treatment options for women with AGCT. 3 Much less is known about germline predisposition to AGCT in comparison to other tumors of the ovary.Germline testing for heritable variants is now recommended for all patients with epithelial ovarian cancers, and both management strategies and treatments have been developed based on testing results. 4For nearly a decade, germline BRCA1/2 pathogenic variants have been established indicators of platinum sensitivity and PARP inhibitor eligibility for these patients. 5Germline testing also presents opportunities for cascade testing and disease prevention within families.][8][9] In contrast, there are currently no targeted therapies or germline testing recommendations for AGCT.A recent study demonstrated that female subjects with the CHEK2 I157T founder allele had increased risk of AGCT. 10 We recently identified two patients with confirmed pathogenic germline variants in ATM and CHEK2 who were undergoing treatment for recurrent AGCT, leading to an interest in examining sequencing data from a larger cohort.A clearer understanding of the role of germline mutations in AGCT oncogenesis and disease biology could create an opportunity for familial cascade testing and risk reduction strategies.We hypothesized that germline-oriented analysis of tumor-only Foundation Medicine testing of a large ACGT cohort could identify potential germline pathogenic variants that may contribute to AGCT risk. 11

| Design, setting, and participants
This is a retrospective cross-sectional study (Figure S1) including all granulosa cell tumor (GCT) specimens submitted for comprehensive genomic profiling (CGP) via FoundationOne®CDx (n = 290), FoundationOne (n = 224), and FoundationOne®Liquid CDx (n = 2) between 2012 and 2022 at Foundation Medicine, Inc. in Cambridge, Massachusetts and Morrisville, North Carolina.FoundationOne, FoundationOne®CDx, and FoundationOne®Liquid CDx are CGP assays for solid tumors, with FoundationOne and FoundationOne®CDx using a tissue sample, and FoundationOne®Liquid CDx using a liquid biopsy.All patients and specimens analyzed are from US-based institutions.Analysis was focused on tumors with a histologic diagnosis of granulosa cell tumor that also harbored a FOXL2 p.C134W mutation characteristic of AGCT.Samples were deduplicated using SNP matching.The study was approved by the Western Institutional Review Board (IRB) and the Medical College of Wisconsin IRB and was reported in accordance with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines. 122.2 | DNA sequencing and processing CGP was performed on formalin-fixed, paraffin-embedded tissue in a Clinical Laboratory Improvement Amendments (CLIA)-certified, College of American Pathologists (CAP)accredited, New York State-approved laboratory, as previously described. 13,14Age, sex, and oncologic diagnosis were abstracted from requisition forms submitted by ordering clinicians.For tissue samples, test requisition forms, pathology reports, and H&E-stained slides were reviewed by a board-certified pathologist to confirm the provided diagnosis.Tumor sections were macrodissected to achieve a minimum of 20% estimated tumor nuclei.
DNA was extracted, quantified, and enriched via adaptor ligation hybrid capture for all coding exons of up to 324 cancer-related genes, depending on the specific bait set, and select introns from genes frequently rearranged in cancer.Sequencing of captured libraries was performed on the Illumina HiSeq platform to a mean exon coverage depth of targeted regions of >500× for tissue samples and >2000× for liquid biopsies.Processed and aligned reads were analyzed for single nucleotide variants, indels, rearrangements, and copy-number alterations, as previously described. 13

| Classification of potential germline pathogenic variants
A pre-defined group of cancer susceptibility genes was evaluated (ATM, BRCA1, BRCA2, BRIP1, CHEK2, MLH1, MSH2, MSH6, PALB2, PMS2, RAD51C, and RAD51D).Assayed genes varied slightly based on the test performed, as the FoundationOne®Liquid CDx and FoundationOne® CDx assays report on more genes than the FoundationOne assay.For example, RAD51C and RAD51D were interrogated in 292 samples due to only being reported on the FoundationOne®Liquid CDx and FoundationOne® CDx assays.The additional 10 genes were interrogated in all 516 cases given their reporting on all three assays.To identify potential germline pathogenic variants, base substitutions and short indels reported by Foundation Medicine testing in these 12 cancer susceptibility genes were first filtered using a sensitivity-optimized variant allele frequency (VAF) threshold of >30%.While germline variants are most likely to have a VAF of 50%, 30%-70% is a commonly accepted threshold. 15ext, variants were filtered based on their classification in ClinVar. 16Variants classified in ClinVar as "Pathogenic," "Pathogenic/Likely Pathogenic," or "Likely Pathogenic" by more than one submitter or by an expert panel were retained.Variants meeting VAF and ClinVar filtering criteria were considered as potential germline pathogenic variants: pathogenic variants of suspected germline origin.
VAF analysis can be used to predict biallelic loss of function of tumor suppressor genes.Mechanisms of biallelic loss of function detectable via VAF analysis include deletion of the second copy and copy neutral loss of heterozygosity (LOH).To determine whether AGCT subjects with potential germline pathogenic DNA repair gene variants fit this model, we compared the VAF of the DNA repair gene mutation to the VAF of the clonal FOXL2 p.C134W mutation.
Predominant genomic patient ancestry was inferred from tissue specimens using a SNP-based classifier that has been previously described. 17

| Clinical and family history analysis of selected patients
Clinical, pathologic, and family histories of two AGCT patients at the Medical College of Wisconsin known to have confirmed germline variants were abstracted via review of medical and genetic counselor documentation.

| Statistical analyses
The odds ratio and two-sided Fisher exact test p-value for GCT risk for patients with the identified potential germline mutations was calculated using gnomAD v2.1 non-cancer subjects as the control group. 18,19Carrier counts were tallied, and the median allele number was used to determine the size of the control population.

| Potential germline pathogenic variants were recurrently observed in DNA repair genes, especially CHEK2
A potential germline pathogenic variant was identified in 6.6% of AGCTs.ATM, BRCA1, BRCA2, CHEK2, PALB2, or PMS2 variant were seen in 30/516 patients, and 3/292 patients had a RAD51C or RAD51D variant (Figure 1; Table 2).The median age of patients with a potential germline pathogenic DNA repair variant was 56, similar to the median of 57 for the cohort overall.The majority of patients were of European ancestry.
Excluded patients (those without a FOXL2 mutation and therefore possibly representing juvenile granulosa cell tumors) were examined as well (Table S2).Of those, 26.9% were found to have a potential germline pathogenic variant in a rare syndrome-associated gene (i.e., DICER1, BAP1, IDH1, and STK11).CHEK2 potential germline pathogenic variants were also seen in 2.5% of this population.

| Loss of heterozygosity was seen in CHEK2 potential pathogenic germline variants in AGCT
Comparisons of the VAF of the DNA repair gene mutation to the VAF of the clonal FOXL2 p.C134W mutation are shown in Figure 2B.Most potential germline pathogenic DNA repair gene variants had VAFs that were slightly higher than the somatic FOXL2 mutation, consistent with monoallelic status for both alterations in the setting of very high median tumor purity of 70% in this cohort. 20In three samples, the FOXL2 VAF was much higher than the DNA repair gene VAF, consistent with loss of heterozygosity (LOH) at the FOXL2 locus, but not at the DNA repair gene locus.Strikingly, 38.9% (7/18) of samples with CHEK2 potential germline pathogenic variants had much higher VAF for the CHEK2 allele compared to the FOXL2 F I G U R E 1 Tile plot depicting selected somatic (upper group) and predicted germline, pathogenic alterations (lower groups) in 516 FOXL2 p.C134W-positive AGCT samples.Each column represents a single patient.6.6% of samples had mutations in DNA repair genes that were predicted to be germline and pathogenic, most commonly in CHEK2 (3.48%, 18/516).*These genes were not tested on all bait sets included in this study.Reported percentages reflect the number of patients tested for the indicated gene.mutation in the same sample, suggesting that CHEK2 is under LOH in those samples.Indeed, examination of these samples showed copy loss of chromosome 22 (example in Figure 2C).Of note, LOH of the remaining CHEK2 allele is also not always observed in breast cancers with confirmed germline CHEK2 variants. 21,22No evidence for LOH was observed for potential germline pathogenic variants in any of the other DNA repair genes investigated.

| Case studies of patients with potential germline pathogenic variants from tumor CGP confirmed on dedicated germline testing
Two patients from this study had clinical history and pedigree information available.The first of these patients was originally diagnosed with stage IC AGCT in 2013 at age 32, and subsequently received multiple surgeries and 5 different lines of treatment.Tumor CGP was performed to investigate additional treatment options, revealing an ATM p.R1875* mutation present at a VAF of 48.2%.Though the patient did not meet contemporaneous NCCN criteria for genetic testing based on her family history, she was referred for follow-up genetic counseling and testing due to her ATM mutation detected on tumor CGP.A detailed family history revealed multiple relatives with cancer, including multiple with breast and prostate cancer (Figure 3A).Dedicated hereditary cancer gene panel testing confirmed that the ATM p.R1875* pathogenic variant was germline in origin.She was treated subsequently with the PARP inhibitor olaparib, on which disease progressed after 13 months.
The second patient was diagnosed with stage IC AGCT in 2007 at age 50.In 2013 she had disease recurrence and underwent secondary cytoreduction; she received multiple surgeries, tumor ablations and 4 lines of systemic therapy.Her 2013 tumor specimen was eventually sent for tumor sequencing in 2018.In 2018, after a CHEK2 p.S428F mutation was identified at a VAF of 13.3%, lower than guidelines often recommend for secondary findings of potential germline pathogenic variants.Genetic counseling was, however, recommended by NCCN guidelines based on her family history, regardless of tumor profiling results (Figure 3B). 23Dedicated germline testing confirmed a germline origin of the CHEK2 p.S428F mutation.Liver metastases were ablated again in 2019 and she underwent a third cytoreductive surgery in 2020.In 2021, she started triplet antihormonal therapy with an anti-androgen, aromatase inhibitor and GnRH agonist.

| DISCUSSION
There is no current recommendation for germline genetic testing in AGCT patients.In this study, we identified a 6.6% (30/516) frequency of potential germline pathogenic variants in this population.Within this group, we identified an elevated incidence of CHEK2 mutations in AGCT patients.A cluster of ATM mutations was also observed in 4/516 patients, but comparison with the frequency in gnomAD non-cancer subjects did not reach statistical significance.Odds ratios were not calculated for all genes due to the infrequent observations of potential pathogenic germline findings in most genes and consequently wide confidence intervals.
CHK2 kinase is a protein in the double-stranded DNA damage response pathway.Carriers of pathogenic germline CHEK2 variants have an increased risk of breast, prostate, colon, kidney, thyroid, and potentially other cancers. 6,24,25Additionally, augmented surveillance for breast and colon cancer is recommended. 24While numerous germline variants within the CHEK2 gene have been described, the two most widely studied are the founder mutations p.T367fs*15 (c.1100delC) and p.I157T. 25 Current cancer risk estimates and management guidelines are based on the CHEK2 1100delC frameshift mutation.The risks associated with missense mutations such as the I157T are lower and the clinical significance remains uncertain. 26 of ovarian cancer patients in the general population).They then examined the prevalence of the two CHEK2 founder mutations (p.I157T and c.1100delC) in AGCT patients and found a positive association with a prevalence ratio of 26.52 (CI95:12.55-56.03)compared to the global population. 10Our findings support this result.We also saw increased CHEK2 VAF in 7 of 18 CHEK2 potential germline pathogenic variants, suggesting some loss of heterozygosity.However, chromosome 22 loss is common in AGCT and seen in 40% of 22 sequenced tumors one study 27,28 , and it is unclear whether this recurrent deletion is related to CHEK2.
While targeted treatments for ovarian cancer patients with CHEK2 mutations have not been F I G U R E 2 Analysis of predicted germline variants DNA repair genes, including CHEK2, GCT samples.(A) Predicted germline, pathogenic CHEK2 alleles were most frequently the common founder mutations I157T and T367fs.(B) Scatter plot comparing FOXL2 C134W VAF versus VAF for predicted germline, pathogenic mutations in selected DNA repair genes.FOXL2 C134W is the key oncogenic driver of most GCTs, and as such, it is presumably clonal in nearly all cases.Interestingly, 39% (7/18) of cases with a CHEK2 alteration exhibited an elevated CHEK2:FOXL2 VAF ratio (red circle), indicating loss of heterozygosity at the CHEK2 locus.(C) Representative copy number plot demonstrating single copy loss and accompanying loss of heterozygosity at the CHEK2 locus on chr 22, resulting in high VAF of the residual pathogenic variant of predicted germline origin.
prospectively confirmed, the presence of a DNA repair deficiency is in accordance with known hallmarks of cancer, and highlights possible utility of platinum or poly (ADP-ribose) polymerase inhibitor (PARPi).PARPi has been explored in multiple cancer types for patients harboring CHEK and ATM mutations and the results have varied. 29For example, while olaparib has been FDA-approved for metastatic CHEK2-and ATM-mutated prostate cancer; therapeutic benefit in these patients was suboptimal in comparison to patients harboring BRCA1/2 mutations. 30For patients with metastatic breast cancer, therapeutic benefit for patients with CHEK2 and ATM mutations when treated with olaparib has not yet been demonstrated, in comparison to those with BRCA1/2 and PALB2 alterations. 31,32Further studies are needed to elucidate the potential efficacy of PARPi or other targeted therapies as treatment for AGCT with DNA repair deficiency.
Current guidelines for patients with epithelial ovarian cancer include recommendations for both somatic and germline testing. 4,6,33However, there are no current guidelines for testing in non-epithelial ovarian cancers.In tumor types in which germline testing is not standard of care, tumor CGP can help identify potential pathogenic germline variants that warrant confirmatory testing.One large study found that while 15.7% of patients who underwent tumor profiling had a potential germline pathogenic variant identified on tumor-only testing, only 40% of these findings were characteristic of the patient's known cancer diagnosis, making the majority of detected inherited risk alleles incidental findings. 34Patients in our study without LOH in their potential germline variant may represent this population.A prior analysis of 104 potential pathogenic germline CHEK2 c.1100delC variants detected on FoundationOne testing found that the variant was reported outside of breast and colorectal cancers 66% of the time (69/104). 35These off-tumor incidental findings are not only common in moderately penetrant genes such as CHEK2, but have been described for highly-penetrant genes such as BRCA1/2, as well. 36The ability of tumor CGP to incidentally identify both on-tumor and off-tumor potential germline pathogenic variants can be seen as complimentary to existing genetic counseling and testing referral algorithms.This has the potential to impact not only patients' therapeutic options and personal cancer screening, but also options for cascade testing and risk stratification of family members.
Strengths of this study included a large sample size for patients with a rare tumor, and comprehensive genomic profiling of the tumor.We were also able to restrict our study sample to a high-confidence set of AGCT patients with a confirmed FOXL2 C134W mutation.Limitations of this study included the likelihood of a biased sample, as patients with advanced or refractory disease may be more likely to undergo tumor CGP.In addition, the potential germline pathogenic variants identified in this exploratory study are suspected germline variants, and dedicated germline testing would be required to confirm germline origin of these pathogenic variant.Outside of the two patient case studies highlighted, it is unknown whether confirmatory germline genetic testing and/or referral to genetic counseling was pursued.Two algorithmic limitations to the filtration of potential pathogenic germline variants are important to consider.First, ClinVar as likely pathogenic or pathogenic mutations in cancer susceptibility genes with VAFs less than 30% are excluded from analysis.Second, rearrangements and copy number alterations are found in a minority of hereditary cancer syndrome patients https:// doi.org/ 10. 1002/ humu.22938 but are not systematically assessed for germline versus somatic origin on this analysis platform and are therefore excluded from this study. 37Given only short variant mutations are included this analysis, other types of potential germline pathogenic variants in the assessed genes are underrepresented.Odds ratio estimates using gnomAD as a control population are confounded by differences in populations and the fact that some subjects who were accrued for non-cancer studies in the gnomAD cohort may in fact have cancer.Other limitations of gnomAD comparisons are that the genomic ancestry is imputed genomic ancestry, but cannot be cross-referenced with patient self-reported information.Imputed ancestry has been shown to be more accurate in Black and White patients than in Mediterranean or Hispanic ethnicity patients. 38ittle has been known about germline variants contributing to AGCT.While the identified incidence of germline variants was lower in FOXL2 C134W-mutated AGCT patients than the epithelial ovarian cancer population, we believe that this association merits further prospective evaluation.If the association is confirmed, women with AGCT could be included in genetic testing referral algorithms.Because organizations such as the NCCN, Society of Gynecologic Oncologists (SGO) and American Society of Clinical Oncology (ASCO) greatly influence genetic testing referral algorithms, this and other similar studies should be taken into consideration when updating practice guidelines pertaining to germline genetic testing.

F I G U R E 3
Pedigree for two patients known to harbor germline mutations and who have documented pedigree information accessible to the investigators.(A) Pedigree for patient with a germline ATM p.R1875* mutation.(B) Pedigree for patient with a germline CHEK2 mutation at p.S428F.
A definitive ovarian cancer risk has not yet been established.Recently, Svajdler, et al. examined 76 female patients with known CHEK2 germline variants, and in those with ovarian cancer, 36% had an AGCT (as compared with 1.3% Clinical summary of AGCT patients.Potential germline variants in DNA repair genes, age, and ancestry of AGCT patients.
T A B L E 1 a Indicates same patient.T A B L E 2